Critical Assessment of the Amerlite Free T4 Assay

Abstract

SUMMARY. The measurement of serum free T4 (FT4) by analogue methods has been severely criticised because the T4 analogue binds to albumin. Amersham have recently introduced a method utilising horseradish peroxidase-labelled-T4 (HRP-T4) designed to overcome this problem and have incorporated it into the Amerlite enhanced luminescence immunoassay system. We have critically evaluated this method for its analytical and clinical validity.Experiments in which anti-albumin was added to normal serum suggested that the HRP-T4 label did not bind to endogenous albumin while the addition of albumin caused no significant change in FT4 concentration. Adding oleic acid up to 5 mmoljL to simulate increased non-esterified fatty acid concentration did not increase the apparent FT4.Serum sampled from subjects independently allocated to clinical groups were compared with an euthyroid group. The untreated hyperthyroid group values were distinctly elevated while the untreated hypothyroid group were appropriately low. Oestrogen therapy, low TBG, familial dysalbuminaemic hyperthyroxinaemia and non-thyroidal illness groups all reflected their euthyroid status, as did pregnancy samples which also showed a tendency to lower values in late pregnancy, consistent with previous observations.In conclusion, the Amerlite FT4 method appears to overcome some of the problems associated with analogue methods. A small survey showed it to be diagnostically valid in a wide variety of clinical states.The measurement of serum free T4 (FT4) concentration, which has long been advocated as a useful test of thyroid function, became more practical as a routine procedure with the development of the antibody-coated tube method! followed by the labelled T4 analogue technique.' It was claimed that the T4 analogue did not compete with any of the bound T4 moieties for T4 antiserum. However, serious objections to this claim were raised both on practical! and theoretical" grounds. The binding of the analogue to serum albumin in particular, caused major inherent problems in the analogue kits.Recently Amersham International pic (Aylesbury, UK) have released a new method for FT4 in association with the Amerlite immunoassay system. With the problems associated with the analogue methods in view, we have assessed the Amerlite FT4 for its methodological soundness and diagnostic performance. Materials and methods ANALYTICAL PROCEDUREThe FT4 method is performed on the Amerlite assay system which uses enhanced luminescence incorporating micro titre well technology with automatic washing, dose response assessment and data reduction. The Amerlite FT4 kit methodology is similar in concept to the analogue-type methods. In the analogue methods, a radioactively labelled analogue of T4 is used which is designed to bind to anti-T4 antibodies with an affinity equal to T4 but not to bind to the serum binding proteins. By contrast, the Amerlite FT4 system uses horseradish peroxidase (HRP)-eonjugated T4 in place of an analogue. The HRP is said to prevent the conjugated T4 from attaching 517